H-Ras induces exuberant de novo dendritic protrusion growth in mature neurons regardless of cell type.

Dendritic protrusions, mainly spines and filopodia, correlate with excitatory synapses in pyramidal neurons (PyNs), but this relationship may not apply universally. We found that ectopic H-Ras expression increased protrusions across various cortical cell types, including layer 2/3 PyNs, parvalbumin (PV)-, and vasoactive intestinal peptide (VIP)-positive interneurons (INs) in the primary motor cortex. The probability of detecting protrusions correlated with local H-Ras activity, indicating its role in protrusion formation. H-Ras overexpression led to high turnover rates by adding protrusions. Two-photon photolysis of glutamate induced de novo spine formation in mature H-Ras expressing neurons, suggesting H-Ras's effect is not limited to early development. In PyNs and PV-INs, but not VIP-INs, spine neck lengths shifted to filopodia-like phenotypes. H-Ras primarily induced filopodia in PyNs and spines in PV- and VIP-INs. Increased protrusions in H-Ras-transfected PyNs lacked key excitatory synaptic proteins and did not affect miniature excitatory postsynaptic currents (mEPSCs), suggesting multifaceted roles beyond excitatory synapses.

Exuberant de novo dendritic spine growth in mature neurons.

Dendritic spines are structural correlates of excitatory synapses maintaining stable synaptic communications. However, this strong spine-synapse relationship was mainly characterized in excitatory pyramidal neurons (PyNs), raising a possibility that inferring synaptic density from dendritic spine number may not be universally applied to all neuronal types. Here we found that the ectopic expression of H-Ras increased dendritic spine numbers regardless of cortical cell types such as layer 2/3 pyramidal neurons (PyNs), parvalbumin (PV)- and vasoactive intestinal peptide (VIP)-positive interneurons (INs) in the primary motor cortex (M1). The probability of detecting dendritic spines was positively correlated with the magnitude of H-Ras activity, suggesting elevated local H-Ras activity is involved in the process of dendritic spine formation. H-Ras overexpression caused high spine turnover rate via adding more spines rather than eliminating them. Two-photon photolysis of glutamate triggered de novo dendritic spine formation in mature neurons, suggesting H-Ras induced spine formation is not restricted to the early development. In PyNs and PV-INs, but not VIP-INs, we observed a shift in average spine neck length towards longer filopodia-like phenotypes. The portion of dendritic spines lacking key excitatory synaptic proteins were significantly increased in H-Ras transfected neurons, suggesting that these increased spines have other distinct functions. High spine density caused by H-Ras did not result in change in the frequency or the amplitude of miniature excitatory postsynaptic currents (mEPSCs). Thus, our results propose that dendritic spines possess more multifaceted functions beyond the morphological proxy of excitatory synapse.